Method and culture device for detecting yeasts and molds

ABSTRACT

A thin film culture device for detecting yeast and mold microorganisms in a sample is provided. The culture device comprises a body comprising a self-supporting substrate having a first major surface and a second major surface; a first adhesive composition disposed on a portion of the first major surface of the substrate; a substantially dry, cold-water-soluble first hydrogel-forming composition adhered to the first adhesive composition; and a plurality of indicator agents. The plurality of indicator agents comprises three indicator agents for detecting distinct glycosidase enzyme activities, an indicator agent for detecting an alkyl esterase enzyme activity, and an indicator agent for detecting a phosphatase enzyme activity, wherein each of the plurality of indicator agents comprises a detectable reporter group. A method of using the culture device is also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/813,897, filed Jul. 30, 2015, now U.S. Pat. No. 9,879,214, which is acontinuation of PCT/US2014/014523, filed Feb. 4, 2014, which claims thebenefit of U.S. patent application Ser. No. 13/775,495, filed Feb. 25,2013, now U.S. Pat. No. 8,921,067 and U.S. Provisional PatentApplication No. 61/760,412, filed Feb. 4, 2013, the disclosures of whichare incorporated by reference in their entirety herein.

BACKGROUND

Yeasts and molds are eukaryotic microorganisms. They are ubiquitous innatural environments, namely, soil, air, water, and plant surfaces.Because of their heterotrophic nature and their ability to adapt to awide range of environmental conditions, these microbes are frequentlyencountered as an expensive nuisance in and on various commoditiesincluding food ingredients, processed foods, beverages, inadequatelycleaned food processing equipment, and food storage facilities. Inaddition, some yeasts and molds possess potential hazard to human andanimal health. For example, numerous molds produce mycotoxins and someyeasts and molds are responsible for human and animal infections.

Yeast or mold contamination in food and other commodities can result insubstantial economic losses for the producer, the processor, and theconsumer. Rapid and accurate determinations of yeast and/or moldcontamination in a commodity (such as, food ingredients, processedfoods, and beverages), are important for the production of high-qualityfood products in the food industry.

Current practices for routine determination of yeasts and molds in afood commodity rely largely on conventional culturing techniques forenumerating viable fungal cells on semi-solid agar media. These methods,although widely accepted, have a number of disadvantages in that theyare, in general, labor intensive and give low reproducibility. Inaddition, a common problem encountered in the traditional methods isthat the spreading type of mycelial growth of certain molds oftenover-runs nearby colonies and prevents accurate enumeration of theviable cells in a sample.

Most importantly, most of these methods require a 5 day incubationperiod at 25° C. before accurate quantitative results can be obtained.The long incubation period of these methods can require that foodproducts be stored for several days, until the presence or concentrationof contaminating yeasts and/or molds is finally known. Thus, there is aneed for improved tests and related materials. If the test procedurescould be simplified, and test results obtained in a shorter period oftime, it would allow manufacturers to release products; thereby reducingstorage costs without sacrificing product quality and integrity.

SUMMARY

The present disclosure generally relates to a culture device and methodfor the detection of yeast or mold microorganisms in a sample. Inparticular, the present disclosure relates to rapid detection of yeastand mold microorganisms in a dry, reconstitutable culture devicecomprising a plurality of indicator agents disposed at highconcentrations in an adhesive composition. The plurality of indicatoragents includes an indicator agent for detecting an alkyl esteraseenzyme activity, an indicator agent for detecting a phosphatase enzymeactivity, and three indicator agents for detecting distinct glycosidaseenzyme activities. Advantageously, the plurality of indicator agentspermits detection and enumeration of a wide variety of slow-growingyeast and mold microorganisms. Even more advantageously, the specificindicator agents, together with the concentrations thereof and the meansfor providing the indicator agents to the microorganisms, permit thedetection and enumeration of the yeast and mold microorganisms in lessthan 120 hours and, preferably, in less than 72 hours. In anyembodiment, the culture device may comprise malt extract.Advantageously, the malt extract acts cooperatively with the pluralityof indicators to provide faster detection of yeast and/or mold coloniesthan otherwise possible.

In one aspect, the present disclosure provides a culture device. Theculture device can comprise a body comprising a self-supportingsubstrate having a first major surface and a second major surface, afirst adhesive composition disposed on a portion of the first majorsurface of the substrate, a substantially dry, cold-water-soluble firsthydrogel-forming composition adhered to the first adhesive composition,and a plurality of indicator agents. The plurality of indicator agentscomprises three indicator agents for detecting distinct glycosidaseenzyme activities, an indicator agent for detecting an alkyl esteraseenzyme activity, and an indicator agent for detecting a phosphataseenzyme activity, wherein each of the plurality of indicator agentscomprises a detectable reporter group. In any embodiment, the culturedevice further can comprise a coversheet attached to the body member.

In any of the above embodiments, the culture device further can comprisea cover sheet attached to the body member. In any embodiment, the coversheet can comprise a first major surface facing the body member; whereinthe culture device further can comprise a second adhesive compositiondisposed on a portion of the first major surface of the cover sheet anda substantially dry, cold-water-soluble second hydrogel-formingcomposition adhered to the second adhesive composition. In any of theabove embodiments, the at least three indicator agents for detectingdistinct glycosidase enzyme activities can include a compound to detectalpha-glucosidase enzyme activity, a compound to beta-glucosidase enzymeactivity, and a compound to beta-galactosidase enzyme activity. In anyof the above embodiments, the at least one nutrient can comprise maltextract. In any of the above embodiments, the culture device further cancomprise a water-insoluble spacer having an aperture, the spacer beingattached to the body member or the coversheet and the entire aperturebeing positioned between the body member and the cover sheet. In any ofthe above embodiments, at least one of the plurality of indicator agentscan be disposed in the first adhesive composition, the second adhesivecomposition, the first hydrogel-forming composition, and/or the secondhydrogel-forming composition. In any of the above embodiments, the firsthydrogel-forming composition or the second hydrogel-forming compositioncan comprise at least one nutrient for growing yeast or moldmicroorganisms.

In another aspect, the present disclosure provides a method of detectingyeast and mold in a sample. The method can comprise contacting a sampleand an aqueous liquid with a gelling agent of a culture device to forman inoculated culture device, incubating the inoculated culture devicefor a period of time, and detecting a yeast or mold colony in theculture device. The culture device comprises a self-supporting substratehaving a first major surface and a second major surface, a firstadhesive composition disposed on a portion of the first major surface ofthe substrate, a cold-water-soluble first hydrogel-forming compositionadhered to the first adhesive composition, and the plurality ofindicator agents. The plurality of indicator agents comprises threeindicator agents for detecting distinct glycosidase enzyme activities,an indicator agent for detecting an alkyl esterase enzyme activity, andan indicator agent for detecting a phosphatase enzyme activity, whereineach of the plurality of indicator agents comprises the detectablereporter group. Optionally, the culture device comprises a coversheetthat includes a second adhesive composition disposed thereon and acold-water-soluble second hydrogel-forming composition adhered to thesecond adhesive composition. The first hydrogel-forming composition orthe second hydrogel-forming composition, if present, comprises thegelling agent and, optionally, at least one nutrient for growing yeastor mold microorganisms. Each of the plurality of indicator agents isdisposed in the first adhesive composition, the second adhesivecomposition, the first hydrogel-forming composition, or the secondhydrogel-forming composition. In any embodiment, the method further cancomprise enumerating yeast or mold colonies present in the inoculatedculture device after incubating the inoculated culture device. In anyembodiment, detecting a yeast or mold colony in the culture device cancomprise detecting in the culture device a presence or an absence of adetectable reporter group of at least one of a plurality of indicatoragents, wherein detecting the presence of the detectable reporter groupis indicative of a presence of a colony of yeast or mold microorganisms.

The term “powder”, as used herein, refers to particulate material of oneor more gelling agents or nutrients having an average diameter suitablefor use in the thin film culture device(s) of the present invention,preferably a diameter of about 10-400 microns more preferably a diameterof about 30-90 microns.

As used herein, “reconstituted medium” refers to a solution or gelformed from the reconstitution of a cold-water-soluble powder with anaqueous liquid.

The term “cold-water-soluble powder”, as used herein, refers to a powderthat forms a gel in room temperature water (e.g., about 18° C. to 24°C.) when combined with an aqueous test sample.

The term “substantially impermeable to microorganisms and water vapor”,as used herein, refers to a cover sheet that prevents undesiredcontamination and hydration of underlying layers of cold-water-solublepowder during shipping, storage, and use of thin film culture device(s),and avoids desiccation of the reconstituted medium, such that thereconstituted medium is suitable to support the growth of microorganismsduring an incubation period.

As used herein, “selective agent” refers to any element, compound, orcomposition that functions to inhibit the growth of one type ofmicroorganism (e.g., a bacterium) relative to another type ofmicroorganism (e.g., a yeast or a mold) and thereby facilitate thegrowth and/or identification of microorganisms grown on the thin filmculture device(s) according to the present disclosure.

The term “yeast”, as used herein, refers to a typically-unicellularfungus of the phylum Ascomycota that reproduces asexually by fissionand/or budding. Yeast includes one or more species existing orco-existing collectively in a test sample. The term “yeasts” also refersto the array of yeasts found, e.g., in a test sample. The terms “yeast”or “yeasts” are not limited to mean any given number of these speciesand are not meant to exclude species which have yet to be discovered butmay later be identified and included in this definition by those ofskill in the art.

The term “mold”, as used herein refers to a microscopic fungus. Thisterm is not limited to mean any given number of these species and is notmeant to exclude species which have yet to be discovered but may laterbe identified and included in this genus by those of skill in the art.

The term “test sample”, as used herein, refers to a component or portiontaken from a food product, a human or animal test subject,pharmaceutical or cosmetic commodity, soil, water, air or otherenvironmental source, or any other source from which a yeast and moldconcentration is to be determined. A test sample may be taken from asource using techniques known to one skilled in the art including, forexample, pouring, pipetting, swabbing, filtering, and contacting. Inaddition, the test sample may be subjected to various sample preparationprocesses known in the art including, for example, blending, stomaching,homogenization, enrichment, selective enrichment, or dilution.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a culture device comprising“an” indicator agent can be interpreted to mean that the culture devicecan comprise “one or more” indicator agents.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The features and advantages of the present invention will be understoodupon consideration of the detailed description of the preferredembodiment as well as the appended claims. These and other features andadvantages of the invention may be described below in connection withvarious illustrative embodiments of the invention.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The figures and the detailed description which follow moreparticularly exemplify illustrative embodiments. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a cross-sectional view of a device of the present disclosure,wherein certain features are shown.

FIG. 2 is a top perspective view, partially in section, of oneembodiment of a culture device according to the present disclosure.

FIG. 3 is a top perspective view of an alternative embodiment of aculture device according to the present disclosure.

FIG. 4 is a cross sectional view of culture device of FIG. 2 taken alongline 4-4.

FIG. 5 is a top view of the culture device of FIG. 2 showing a gridpattern printed on the microporous membrane.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. Unless specified or limitedotherwise, the terms “connected” and “coupled” and variations thereofare used broadly and encompass both direct and indirect connections andcouplings. Further, “connected” and “coupled” are not restricted tophysical or mechanical connections or couplings. It is to be understoodthat other embodiments may be utilized and structural or logical changesmay be made without departing from the scope of the present disclosure.Furthermore, terms such as “front,” “rear,” “top,” “bottom,” and thelike are only used to describe elements as they relate to one another,but are in no way meant to recite specific orientations of theapparatus, to indicate or imply necessary or required orientations ofthe apparatus, or to specify how the invention described herein will beused, mounted, displayed, or positioned in use.

Yeasts and molds are ubiquitous eukaryotic microorganisms that remainviable in certain (e.g., low water activity, low pH) environments thatare detrimental to most other unicellular microorganisms. Classified asfood spoilage indicator organisms, tests for yeasts and molds constituteabout 14% of all routinely conducted indicator-organism testing becauseyeast and mold microorganisms are frequently encountered as an expensivenuisance in and on various commodities (e.g., food ingredients,processed foods, beverages), inadequately cleaned food processingequipment, and food storage facilities. In addition, some yeasts andmolds have clinical relevance as causative agents of human and animalinfections.

Traditional testing for yeast and molds typically involve culturing themicroorganisms on agar media or in thin-film culture devices such as a3M PETRIFILM Yeast & Mold Count Plate (available from 3M Company, St.Paul, Minn.). Thin film culture devices for detecting yeast and mold ina sample are described, for example, in U.S. Pat. No. 5,089,413, whichis incorporated herein by reference in its entirety. The traditionaltesting methods involve incubating the test plates for a period of up to5-7 days to get accurate counts. This relatively lengthy time to resultcan cause food manufacturers to hold the food product prior for up toseven days in order to determine whether the food product contains ayeast or mold microorganism that may adversely affect the quality and/orsafety of the food product during normal conditions of storage.

Some culture media, including the culture medium used in 3M PETRIFILMYeast & Mold Count plates, include at least one indicator reagent (e.g.,5-bromo-4-chloro-3-indolylphosphate sodium salt) that is converted byyeast or mold microorganisms to a detectable (e.g., detectable by lightabsorption, reflectance, and/or fluorescence) product. U.S. Pat. No.6,387,650; which is incorporated herein by reference in its entirety;discloses compositions for detecting yeast and fungi in a test sample,wherein the compositions comprise three enzyme substrates that cause orproduce an identical type of detectable signal (e.g., fluorescence) whenhydrolyzed by a corresponding enzyme activity found in themicroorganisms. One enzyme substrate is hydrolyzed by a glycosidaseenzyme activity, a second enzyme substrate is hydrolyzed by a peptidaseenzyme activity and the third enzyme substrate is hydrolyzed by aphosphatase enzyme activity. In the patent, Townsend and Chen furtherdescribe the use of media containing the indicator reagents in a culturedevice that permits the calculation of a most probable number (MPN) ofmicroorganisms in the test sample.

Chen and Gu (U.S. Pat. No. 5,854,011; which is incorporated herein byreference in its entirety) describe a method and compositions fordetecting yeast and/or molds in a sample wherein the compositionincludes an aminopeptidase enzyme substrate and an inhibitor ofaminopeptidase enzyme activity in an amount effective to inhibitaminopeptidase activity that is endogenous to the test sample. InExample 1, Chen and Gu conclude that, because they were found in ≤64% ofthe microorganisms tested, phosphatase, β-glucosidase, and α-glucosidaseenzymes are unsuitable to be used as the target enzyme for detectingtotal yeasts and molds in a sample.

As evidenced by Chen and Gu, it is difficult to find an enzyme substratethat can detect the wide variety of yeasts and molds that are found innature. The inventive article and method of the present disclosureprovide detection of a variety of yeast and mold. In addition, theinventive article and method can provide rapid detection of the yeastand mold microorganisms. In some embodiments, the investigators havediscovered a composition comprising a unique combination of indicatorsin a nutrient medium comprising malt extract. In a culture device of thepresent disclosure, the composition is suitable for the rapid detectionof a variety of yeast and mold microorganisms.

In one aspect, the present disclosure provides a culture device fordetecting yeast and mold microorganisms in a sample. The components ofthe culture device, when contacted with an aqueous liquid, can actcooperatively to form an aqueous culture medium that is used tocultivate yeast and mold (e.g., filamentous fungi) microorganisms. Inany embodiment, the culture medium of the present disclosure can be amixture which comprises all or substantially all of the nutrientsnecessary to support the growth of yeasts and molds. In someembodiments, one or more nutrients to support the growth of yeast andmold microorganisms may be provided in the sample.

The culture device of the present disclosure provides improved detection(i.e., reduced time to detection, more inclusive detection of yeast andmold microorganisms within a specified incubation period) compared toother devices and methods known in the art. In some aspects, a culturedevice of the present disclosure is related to thin film culture devicesdisclosed in U.S. Pat. Nos. 4,565,783; 5,089,413; and 5,681,712; whichare all incorporated herein by reference in their entirety.

Suitable samples for use with the inventive culture device can beobtained or derived from a variety of sources. The term “source” isgenerally used to refer to the food or nonfood desired to be tested formicroorganisms. The source can be a solid, a liquid, a semi-solid, agelatinous material, gas (e.g., air), and combinations thereof. In someembodiments, the source can be provided by a capture element (e.g., afilter membrane, swab, fabric, or sponge) that was used, for example, tocollect the source from a surface of interest or from air. In someembodiments, a sample liquid can include the capture element, which canbe further broken apart (e.g., during an agitation or dissolutionprocess) to enhance retrieval of the source and any microorganism ofinterest. The surface of interest can include at least a portion of avariety of surfaces, including, but not limited to, walls (includingdoors), floors, ceilings, drains, refrigeration systems, ducts (e.g.,air ducts), vents, toilet seats, handles, doorknobs, handrails,countertops, tabletops, eating surfaces (e.g., trays, dishes, etc.),working surfaces, equipment surfaces, clothing, etc., and combinationsthereof. All or a portion of the source can be used in the method. Whena portion of the source is used, this can sometimes be referred to as a“sample” of the source. However, the term “sample” is generally usedherein to refer to the portion of volume or mass of material that isobtained from the source and is introduced into a test device for thedetection of microorganisms.

The term “food” is generally used to refer to a solid, liquid (e.g.,including, but not limited to, solutions, dispersions, emulsions,suspensions, etc., and combinations thereof) and/or semi-solidcomestible composition. Examples of foods include, but are not limitedto, meats, poultry, eggs, fish, seafood, vegetables, fruits, preparedfoods (e.g., soups, sauces, pastes), grain products (e.g., flour,cereals, breads), canned foods, milk, other dairy products (e.g.,cheese, yogurt, sour cream), fats, oils, desserts, condiments, spices,pastas, beverages, water, animal feed, other suitable comestiblematerials, and combinations thereof.

With reference to FIG. 1, a device of the present disclosure is shown asbody member 10 with three salient features: waterproof substrate 12,air-permeable membrane 14, and substantially dry, cold-water-solublefirst hydrogel-forming composition 22. Although these can be arranged inany suitable relationship, FIG. 1 illustrates a preferred arrangement ofthese components, wherein air permeable membrane 14 is fixed to andcovers at least the growth region (not shown) of the top surface ofsubstrate 12. First hydrogel-forming composition 22 is fixed to andcovers at least the growth region of the top surface of membrane 14.Cover means (coversheet 18) for covering the first hydrogel-formingcomposition 22 during shipping, storage, and incubation, is also shownin FIG. 1 as being attached in a hinge-like fashion along one edge ofbody member 10. Cover means are optional but preferred in devices of thepresent disclosure. Suitable substrates, first hydrogel-formingcomposition and cover means include those described in U.S. Pat. No.4,565,783, which is incorporated herein by reference in its entirety.

Substrate 12 can be a relatively stiff film (e.g., polyester,polypropylene or polystyrene) or a relatively stiff paper or cardboardhaving a water-resistant coating thereon, which will not absorb orotherwise be affected by water. Polyester films approximately 100μ, to180μ, thick, polypropylene films approximately 100μ, to 200μ, thick, andpolystyrene films approximately 300μ, to 380μ thick are nonlimitingexamples of suitable materials for the substrate 12. The substrate 12may be either transparent or opaque, depending on whether one wishes toview microorganism colonies through the substrate. To facilitate thecounting of microorganism colonies, the substrate 12 optionally can havea grid pattern (e.g., squares, not shown) printed thereon.

Air-permeable membrane 14 allows an adequate supply of air to firsthydrogel-forming composition 22 when coversheet 18 is in place after thedevice is inoculated. In so doing, membrane 14 is useful for supportinggrowth of aerobic microorganisms in the device. By virtue of the airpermeability of the membrane and the membrane being substantiallyexposed at its edge(s) to air, air is able to pass into the edge(s) ofthe membrane, horizontally through the membrane, and into the medium.Horizontal passage of air for a particular membrane is most convenientlyestimated by evaluating the vertical air permeability of the membrane(i.e., permeability in a direction normal to the top and bottom surfacesof the membrane). Suitable air permeable membrane 14 materials,including microporous films and microporous non-woven webs of syntheticor natural materials, are described in U.S. Pat. No. 5,089,413, which isincorporated herein by reference in its entirety. A nonlimiting exampleof a preferred membrane material is a microporous polyolefin film(TREDEGAR EXXAIRE film, Tredegar Film Products, Richmond, Va.).

In any embodiment, the membrane 14 has a visible square grid patternprinted upon it, as shown in FIG. 5, to facilitate the counting ofmicroorganism colonies. A device of the present disclosure can beprepared using a variety of techniques. Generally, a device can be madeby hand or with common laboratory equipment as described in detailbelow.

FIGS. 2 and 4 illustrate a device in accordance with the presentdisclosure. Device 28 includes a body member 10′ having a water-proofsubstrate 12 with a top surface and a bottom surface. The bottom surfaceof membrane 14 is fixed to (e.g., fixed with an adhesive or otherwiseattached to) at least the growth region (not shown) of the top surfaceof substrate 12. Preferably, the top surface of substrate 12 is coatedwith adhesive layer 30, which is used to fix membrane 14. Adhesive layer30 is preferably pressure-sensitive, insoluble in water, andsubstantially non-inhibitory to the growth of the intendedmicroorganisms, as described herein. Preferred adhesives include thosediscussed below in connection with first adhesive composition 20 andsecond adhesive composition 20′. Often, suitable substrates areavailable already coated with a suitable adhesive. If one desires,however, a suitable substrate can be selected and coated (e.g., using aknife coater) with a suitable adhesive.

The method of fixing membrane 14 to substrate 12 will depend on thenature of adhesive layer 30. If adhesive layer 30 is pressure sensitivefor instance, membrane 14 can be placed on adhesive layer 30, presseddown, and thereby adhered in place.

Referring to FIGS. 2 and 3, substrate 12 is coated on a portion of itsfirst surface with a layer of first adhesive composition 20. In anyembodiment, the first adhesive composition 20 may comprise one or moreindicator agents (not shown) and/or one or more selective agents (notshown) as described herein. Adhered to the first adhesive composition 20is a substantially dry, cold-water-soluble first hydrogel-formingcomposition 22 which, optionally, includes a powdered nutrient 16. Thus,the thickness of the first adhesive composition 20 preferably should beless than the diameter of the particles of the powdered gelling agentand/or nutrients that make up the cold-water-soluble firsthydrogel-forming composition 22. A uniform monolayer ofcold-water-soluble first hydrogel-forming composition 22 is desired withsufficient surface area exposed for hydration.

An adhered powder medium, illustrated in FIGS. 2 and 3, is prepared andfixed by first forming a layer of first adhesive composition 20 on atleast the growth region of the top surface of membrane 14. The adhesiveof the first adhesive composition 20 is preferably pressure-sensitive,insoluble in water, and substantially non-inhibitory to the growth ofthe intended microorganisms. Preferably, first adhesive composition 20is also sufficiently transparent when wet to enable viewing of microbialcolonies.

Attached to the substrate 12 is optional coversheet 18. In anyembodiment, cover sheet 18 can be affixed to one edge of substrate 12(e.g., via heat sealing or a double-sided tape). Cover sheet 18 istranslucent or preferably transparent to facilitate counting of themicroorganism colonies, and is substantially impermeable to bothmicroorganisms and water vapor. Generally, cover sheet 18 will have thesame properties, such as transparency and preferred waterimpermeability, as substrate 12. Furthermore, cover sheet 18 can havepatterns imprinted thereon, such as square grid pattern, or a mask-edge(not shown) to aid in the counting of microorganism colonies, to providea target for placement of the aqueous test sample, and/or for aestheticreasons. Cover sheet 18 can be selected to provide an amount of oxygentransmission necessary for yeast and mold microorganisms, some of whichrequire relatively oxygen-rich environments for optimal growthconditions. Suitable cover sheet materials are disclosed in U.S. Pat.No. 5,681,712.

Cover means (e.g., coversheet 18) can be free of any coating, or can becoated, e.g., on the surface facing the dry medium with a layer ofpressure-sensitive adhesive, in order to facilitate sealing of the covermeans over the medium. Furthermore, coversheet 18 can optionally becoated on the surface facing the first hydrogel-forming composition 22with layers of second adhesive composition 20′ and secondhydrogel-forming composition 22′, that are the same as or different fromfirst adhesive composition 20 and first hydrogel-forming composition 22,respectively. Coatings on coversheet 18 can cover the entire surfacefacing the first hydrogel-forming composition 22, but preferably coverat least the part of the surface that is intended to cover the growthregion of the culture device. Such coated coversheets are particularlypreferred when it is desired to provide a device with more gelling agentthan can be incorporated in the first hydrogel-forming compositionalone.

Coversheet 18 is preferably adhered in a hinge-like fashion along oneedge of spacer 24, and is optionally coated with a layer of secondadhesive composition 20′ and second hydrogel-forming composition 22′. Inany embodiment, the second adhesive composition can comprise one or more(e.g., all) of the plurality of indicator agents used to detect yeastand/or mold microorganisms. Alternatively, coversheet 18 can be adhereddirectly to the substrate 12 as illustrated in FIG. 3.

If a culture device (not shown) of the present disclosure does notinclude a coversheet attached to the substrate, the culture deviceshould be stored and incubated in a container (e.g., a petri dish) inorder to prevent contamination and/or desiccation of the device andsample before and after inoculation.

Also shown in FIG. 2 is an optional second adhesive composition 20′disposed on at least a portion of coversheet 18; a substantially dry,cold-water-soluble second hydrogel-forming composition 22′ disposed onsecond adhesive composition 20′; and an optional spacer 24. Whenhydrated with an aqueous liquid (e.g., a liquid sample and/or an aqueoussuspending medium, such as water or a buffer), a gelling agent presentin the first hydrogel-forming composition 22 and/or secondhydrogel-forming composition 22′ forms a hydrogel.

As depicted in FIGS. 2 and 4, the culture device can include a spacer 24attached (e.g., via heat bonding or a pressure-sensitive adhesive) to afirst surface of substrate 12, the first adhesive composition 20, and/orthe first hydrogel-forming composition 22. The spacer 24 comprises anaperture (e.g., circular aperture 26) cut through the center to exposethe first hydrogel-forming composition 22. The walls of aperture 26provide a well of predetermined size and shape to confine the hydrogelfollowing hydration of the first hydrogel-forming composition 22 with anaqueous liquid. The aperture 26 generally delineates a growth area ofthe culture device. Spacer 24 should be thick enough to form a well ofthe desired volume, e.g., 1, 2 or 3 milliliters. Closed cellpolyethylene foam is a preferred material for spacer 24, but anymaterial which is hydrophobic (non-wetting), inert to microorganisms,and capable of withstanding sterilization may be used. In someembodiments (not shown), the spacer can comprise a plurality ofapertures (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 apertures),each of which can be inoculated with a distinct sample.

Suitable materials for the spacer member are any solid non-inhibitorynatural or synthetic substance which is readily available in sheet formbut is not a microorganism growth site. Polyethylene, polypropylene,polyethylene terephthalate and polystyrene are a few examples ofsuitable synthetic materials. In particular, relatively inexpensivecommercially available polystyrene foams and polyethylene foams arepreferred.

Spacer 24 can include relatively thick designs, such as those describedin U.S. Pat. No. 5,681,712. One purpose of the thicker (e.g., at leastabout 0.5 mm thick, about 1 mm thick, about 1.5 mm thick and about 2 mmthick) apertured spacer 24 is to locate and protect membranes (e.g.microporous filter membranes, not shown) placed in the aperture 26 ofthe spacer 24. Another purpose of the thicker spacer 24 is to reduce orprevent contact by cover sheet 18 with the growing colonies ofmicroorganisms (i.e., provide a “head space” between the growth surfaceand the cover sheet 18, which can also provide increased aeration forgrowing colonies of microorganisms).

The first adhesive composition 20 and, if present, second adhesivecomposition 20′ preferably is a pressure sensitive adhesive. Morepreferably, the adhesive is a pressure-sensitive adhesive such as awater-insoluble adhesive comprising a copolymer of an alkyl acrylatemonomer and an alkyl amide monomer. Preferably the weight ratio of alkylacrylate monomer to alkyl amide monomer in these copolymers is fromabout 90:10 to 99:1, more preferably 94:6 to 98:2. The alkyl acrylatemonomer comprises a lower alkyl (C2 to C10) monomer of acrylic acid,including, for example, isooctyl acrylate (IOA), 2-ethylhexyl acrylate,butyl acrylate, ethyl acrylate, isoamyl acrylate, and mixtures thereof,while the alkyl amide monomer can comprise, without limitation,acrylamide (ACM), methacrylamide, N-vinylpyrrolidone (NVP),N-vinylcaprolactam (NVCL), N-vinyl-2-piperidine, N-(mono- or di-loweralkyl (C2 to C5))(meth)acrylamides, N-methyl(meth)acrylamide,N,N-dimethyl(meth) acrylamides, or mixtures thereof.

In any embodiment, the first adhesive composition 20 and/or secondadhesive composition 20′ may comprise an indicator agent (not shown)and/or selective agent. In any embodiment, the indicator agent may bedissolved in an organic solvent (e.g., methanol) and blended with theadhesive composition before applying the composition to the substrate 12and/or coversheet 18. In any embodiment, the first adhesive composition20 and/or second adhesive composition 20′ may include a plurality ofindicator agents. In any embodiment the first adhesive composition 20and second adhesive composition 20′ each may include an identicalindicator agent. In any embodiment the first adhesive composition 20 andsecond adhesive composition 20′ each may include an indicator agent orselective that is not included in the other adhesive composition.

In any embodiment, one or more indicator agent of the present disclosurecan be disposed as a powder (or an agglomerated powder) in the firsthydrogel-forming composition 22 and/or second hydrogel-formingcomposition 22′ disclosed herein.

In any embodiment, a culture device of the present disclosure comprisesa plurality of indicator agents (e.g., enzyme substrates), eachindicator agent comprising a reporter group (e.g., a fluorogenic groupor chromogenic group) that permits detection of a reaction between theindicator agent and a biological activity (i.e., an enzyme activityassociated with a yeast or mold microorganisms) and the indicator agent.In any embodiment, the plurality of indicator agents can comprise fiveindicator agents. In any embodiment, the five indicator agents caninclude three agents for detecting three distinct glycosidase enzymeactivities, an indicator agent for detecting an esterase enzyme activity(e.g., an alkyl esterase enzyme activity), and an indicator agent fordetecting a phosphatase enzyme activity. The culture medium furthercomprises a gelling agent. In any embodiment, the plurality of indicatoragents does not include an indicator agent for detecting aminopeptidaseenzyme activity.

Yeast and mold microorganisms produce one or more of a variety ofglycosidase enzyme activities, each glycosidase enzyme activity beingcapable of reacting with an indicator reagent to produce a detectableproduct. Tables 1 and 2 list nonlimiting examples of indicator agentsthat may react with a corresponding enzyme activity, if present, withinor proximate a colony of yeast or mold microorganisms.

TABLE 1 Indicator agents for detecting glycosidase enzyme activities.4-Methylumbelliferyl-N- 4-Methylumbelliferyl-β-D- 4-Nitrophenyl-β-D-acetate-β-D-galactosaminide xylose fucopyranoside4-Methylumbelliferyl-N- 6-Bromo-2-naphthyl-N-acetyl- 2-Nitrophenyl-β-D-acetate-β-D-glucosaminide β-D-glucosaminide thiogalactopyranoside2′-(4-Methylumbellifelyl-α-D- 6-Bromo-2-naphthyl-α-D-Phenolphthalein-mono-β-D- N-acetyl-neuraminic acid glucopyranosidegalactopyranoside Sodium salt 4-Methylumbelliferyl-α-L-6-Bromo-2-naphthyl-β-D- 5-Bromo-4-chloro-3-Indolyl-N- arabinopyranosidexylopyranoside acetyl-β-D-galactosaminide 4-Methylumbelliferyl-β-D-Naphthol AS-BI-β-L- 5-Bromo-4-chloro-3-Indolyl-β- cellobiopyranosidefucopyranoside D-fucopyranoside 4-Methylumbelliferyl-β-D-1-Naphthyl-α-D- Indoxyl-β-D-galactoside fucoside galactopyranoside4-Methylumbelliferyl-α-D- 2-Nitrophenyl-N-acetyl-α-D- 4-Nitrophenyl-α-L-mannoside galactosaminide fucopyranoside 4-Methylumbelliferyl-6-sulfo-4-Nitrophenyl-β-D-cellobioside 4-Nitrophenyl-β-L-N-acetyl-β-D-glucosaminide fucopyranoside 4-Methylumbelliferyl-β-D-6-Bromo-2-naphthyl-β-D- 2-Nitrophenyl-α-D- cellotriose galactosidegalactopyranoside 4-Methylumbelliferyl-β-D- 6-Bromo-2-naphthyl-β-D-2-Nitrophenyl-β-D- N,N′-diacetyl-chitobioside glucopyranosidegalactopyranoside 4-Methylumbelliferyl-α-L- 6-Bromo-2-naphthyl-β-D-3-Nitrophenyl-α-D- fucoside glucuronide galactopyranoside4-Methylumbelliferyl-β-L- 2-Chloro-4-nitrophenyl-N- 3-Nitrophenyl-β-D-fucoside acetyl-β-D-glucosaminide galactopyranoside4-Methylumbelliferyl-α-D- 2-Chloro-4-nitrophenyl-β-D- 4-Nitrophenyl-α-D-galactoside cellobioside galactopyranoside 4-Methylumbelliferyl-β-D-2-Chloro-4-nitrophenyl-β-D- 4-Nitrophenyl-β-D- galactosidexylopyranoside galactopyranoside 4-Methylumbelliferyl-β-D-β-Hydroxyquinoline-β-D- 4-Nitropheny1-β-D- galactoside-6-phosphateglucuronide glucuronide Ammonium salt 4-Methylumbelliferyl-α-D- NaphtholAS--BI-β-D- 4-Nitrophenyl-α-D- glucoside galactopyranosideglucopyranoside 4-Methylumbelliferyl-β-D- Naphthol AS--BI-β-D-4-Nitrophenyl-β-D- glucoside galactosaminide glucopyranoside4-Methylumbelliferyl-β-D- Naphthol AS--BI- 4-Nitrophenyl-β-D-glucuronideglucuronide glucopyranoside 4-Methylumbelliferyl-β-D- NaphtholAS--BI-β-D- 2-Nitrophenyl-β-D-glucuronide N,N′,N′-triacetylchitotrioseglucuronic acid 5-Bromo-4-chloro-3-Indolyl-α- 1-Naphthyl-β-D-4-Nitrophenyl-β-D-glucuronide D-galactopyranoside galactopyranoside4-Nitrophenyl-N-acetyl-1-thio- 2-Naphthyl-β-D- 4-Nitrophenyl-β-D-β-D-glucosaminide galactopyranoside thiogalactopyranoside4-Nitrophenyl-α-L- 1-Naphthyl-β-D-glucuronide 4-Nitrophenyl-β-D-arabinopyranoside thioglucopyranoside 3-Nitrophenyl-β-D-4-Nitropheny1-N-acetyl-α-D- Phenolphthalein-β-D- fucopyranosidegalactosaminide glucuronic acid Sodium salt 4-Nitrophenyl-α-D-4-Nitropheny1-N-acetyl-β-D- Phenyl-N-acetyl-α-D- fucopyranosidegalactosaminide glucosaminide 5-Bromo-4-chloro-3-Indolyl-β-4-Nitrophenyl-N-acetyl-α-D- Phenylethyl-β-D-galactosideD-glucopyranoside glucosaminide 5-Bromo-4-chloro-3-Indolyl-β-4-Nitrophenyl-N-acetyl-β-D- Phenyl-β-D-galactoside D-glucuronic acidglucosaminide Cyclohexylammonium salt 5-Bromo-4-chloro-3-Indolyl-β-Indoxyl-β-D-glucoside Phenyl-α-D-glucoside D-glucuronic acid Sodium salt5-Bromo-4-chloro-3-Indolyl-α- Indoxyl-β-D-glucuronic acid5-Bromo-4-chloro-3-Indolyl-N- D-mannopyranoside Cyclohexylammonium saltacetyl-β-D-glucosaminide ALDOL 467 β-D- ALDOL 470 α-D- ALDOL 470 β-D-glucosaminide glucopyranoside galactopyranoside ALDOL 518 β-D- ALDOL 467β-D- ALDOL 458 β-D- galactopyranoside galactopyranosidegalactopyranoside

Yeast and mold microorganisms produce a variety of esterase enzymeactivities including, for example, alkyl esterase (e.g., fatty acidalkyl esterase) enzyme activities and phosphatase (e.g., phosphoricmonoester hydrolase) enzyme activities. Table 2 lists nonlimitingexamples of indicator agents that may react with a correspondingesterase enzyme activity, if present, within or proximate a colony ofyeast or mold microorganisms.

TABLE 2 Indicator agents for detecting alkyl esterase phosphatase enzymeactivities. Alkyl esterase enzyme substrates Phosphatase enzymesubstrates 4-Methylumbelliferyl-acetateBis(4-methylumbellifelyl)-phosphate 4-Methylumbelliferyl-butyrateBis(4-methylumbellifelyl)-phosphate Sodium salt4-Methylumbelliferyl-laurate 4-Methylumbelliferyl-phosphate (free acid)4-Methylumbelliferyl-nonaoate 4-Methylumbelliferyl-phosphateDicyclohexylammonium salt 4-Methylumbelliferyl-oleate4-Methylumbelliferyl-phophate Disodium salt4-Methylumbelliferyl-palmitate Bis(4-nitrophenyl)phosphate Sodium salt4-Methylumbelliferyl-propionate Naphthol AS-phosphate4-Methylumbelliferyl-stearate Naphthol AS-phosphate Sodium salt6-Bromo-2-naphthyl acetate 1-Naphthylphosphate Disodium salt NaphtholAS-acetate 2-Naphthylphosphate Disodium salt Naphthol AS-nonanoate2-Naphthylphosphate Sodium salt 1-Naphthylbutyrate 2-NaphthylphosphateSodium salt 2-Naphthylbutyrate 1-Naphthylphosphate Sodium salt1-Naphthylcaprylate Phenolphthalein diphosphate 2-NaphthylcaprylatePhenolphthalein diphosphate Tetrasodium salt 2-Nitrophenyl-acetate5-Bromo-4-chloro-3-Indolyl-phosphate Disodium salt 4-Nitrophenyl-acetate5-Bromo-4-chloro-3-Indolyl-phosphate Potassium salt2-Nitrophenyl-butyrate 5-Bromo-4-chloro-3-Indolyl-phosphate p-Toluidinesalt 4-Nitrophenyl-butyrate 3-Indoxyl-phosphate Di(2-amino-2-methyl-1,3-propanediol) salt 4-Nitrophenyl-caprate 3-Indoxyl-phosphate Disodiumsalt 4-Nitrophenyl-caproate 3-Indoxyl-phosphate p-Toluidine salt3-Nitrophenyl-caprylate ALDOL 470 phosphate, disodium salt4-Nitrophenyl-caprylate ALDOL 458 phosphate, disodium salt2-Nitrophenyl-myristate 4-Nitrophenyl-myristate 2-Nitrophenyl-palmitate4-Nitrophenyl-palmitate 4-Nitrophenyl-propionate 4-Nitrophenyl-stearate5-Bromo-4-chloro-3-Indolyl-acetate 5-Bromo-4-chloro-3-Indolyl-butyrate5-Bromo-4-chloro-3-Indolyl-caprylate ALDOL 515 acetate ALDOL 470 acetateALDOL 470 butyrate ALDOL 470 nanoate ALDOL 458 acetate

In a preferred embodiment, the culture device of the present disclosurecomprises an indicator agent for detecting α-glucosidase enzymeactivity, an indicator agent for detecting β-glucosidase enzymeactivity, and an indicator agent for detecting β-galactosidase enzymeactivity. In any embodiment, all three of the aforementioned indicatoragents comprise similar or identical reporter groups. In a particularlypreferred embodiment, the culture device of the present disclosurecomprises 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, and5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

According to the present disclosure, the indicator agents can beprovided in the culture device as a powder or agglomerated powder (i.e.,as a component of the cold-water-soluble first hydrogel-formingcomposition and/or the cold-water-soluble second hydrogel-formingcomposition described herein) or as a part of the first adhesivecomposition and/or second adhesive composition described herein.Advantageously, when provided in at least one of the adhesivecompositions, the indicator agents can be uniformly distributed withinthe growth area of the culture device and can be provided in theadhesive composition at a very high concentration. Without being boundby theory, it is thought the indicator agents efficiently partition fromthe relatively hydrophobic adhesive composition into the relativelyhydrophobic reconstituted gel, thereby providing consistent, uniformconcentrations of the indicator agents to react with yeast or moldmicroorganisms, if present in the sample, in the culture device.

In any embodiment, the first and/or second adhesive composition cancomprise five indicator agents. The five indicator agents can comprise5-Bromo-4-chloro-3-indolyl acetate,5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside,5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside,5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside, and5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt. As shown in Table13 below, the investigators have found this particular combination ofindicator agents has the surprising effect of providing for thedetection of at least one organism (e.g., a microorganism belonging tothe genus Botrytis) that cannot otherwise be detected using a similarculture device having only one of the aforementioned indicator agents,indicating a possible synergistic effect of the combination.

In any embodiment, the first or second adhesive composition can compriseabout 0.05-1.0 weight percent 5-Bromo-4-chloro-3-indolyl acetate, about0.05-1.0 weight percent5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 0.05-1.0 weightpercent 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, about 0.05-1.0weight percent 5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside and/orabout 0.05-1.0 weight percent 5-Bromo-4-chloro-3-indolyl phosphatep-toluidine salt.

In any embodiment, the first or second adhesive composition can compriseabout 0.18-0.5 weight percent 5-Bromo-4-chloro-3-indolyl acetate. In anyembodiment, the first or second adhesive composition can comprise about0.34-0.72 weight percent5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside. In any embodiment, thefirst or second adhesive composition can comprise about 0.34-0.72 weightpercent 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside. In anyembodiment, the first or second adhesive composition can comprise about0.34-0.72 weight percent 5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside.In any embodiment, the first or second adhesive composition can compriseabout 0.36-0.76 weight percent 5-Bromo-4-chloro-3-indolyl phosphatep-toluidine salt.

In any embodiment, the first or second adhesive composition can compriseabout 0.18-0.5 weight percent 5-Bromo-4-chloro-3-indolyl acetate, about0.34-0.72 weight percent5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, about 0.34-0.72 weightpercent 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, about 0.34-0.72weight percent 5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside, and about0.36-0.76 weight percent 5-Bromo-4-chloro-3-indolyl phosphatep-toluidine salt.

Adhered to the first adhesive composition 20 is a cold-water-solublefirst hydrogel-forming composition 22. The first hydrogel-formingcomposition 22 comprises at least one cold water soluble gelling agent.As indicated above, the dry medium can contain gelling agent only, andno nutrient. Optionally, in any embodiment, the first hydrogel-formingcomposition 22 also may comprise a nutrient for growing yeast or moldmicroorganisms. Suitable gelling agents for use in firsthydrogel-forming composition 22 include cold-water-soluble natural andsynthetic gelling agents. Non-limiting examples of suitable naturalgelling agents include algin, carboxymethyl cellulose, hydroxyethylcellulose, locust bean gum, xanthan gum. Suitable synthetic gellingagents include, for example polyacrylamide. Combinations of naturaland/or synthetic gelling agents are contemplated. Preferred gellingagents include locust bean gum and xanthan gum, these gelling agentsbeing useful individually or, in any embodiment, in combination with oneanother.

First hydrogel-forming composition 22 can contain the componentsdiscussed above in connection with dry media. Preferably, when gellingagent is included in first hydrogel-forming composition 22, it isincluded in an amount such that a predetermined quantity of water or anaqueous sample, e.g., 1 to 3 ml, placed on the medium will form areconstituted medium having a suitable viscosity, e.g., about 1500 cpsor more when measured at 60 rpm with a Brookfield Model L VF viscometerat 25° C. Media of this viscosity allow convenient handling and stackingof the devices during incubation and provide for distinct colonyformation in the medium. For instance, 0.025 g to 0.050 g of powderedguar gum spread substantially uniformly over a surface area of 20.3 cm²will provide a sufficiently viscous medium when reconstituted with 1 to3 ml of an aqueous sample. The size of the powder particles can be usedto control the coating weight per unit area. For example, underconditions where a 100 mesh guar gum coats to a weight of about 0.05g/20.3 cm², a 400 mesh guar gum coats to a weight of about 0.025 g/20.3cm². The first hydrogel-forming composition can be applied to the firstadhesive composition 20 using methods described in U.S. Pat. No.5,089,413.

As indicated above, the dry medium can contain gelling agent only, andno nutrient. Before the addition of an aqueous sample liquid (e.g., aliquid sample suspected of containing yeast or mold microorganisms) tothe culture device, the user can add nutrients tailored to the type ofmicroorganisms to be grown. For example, dry powdered nutrients can besuspended in a rapidly-evaporating liquid such as ethanol or a volatilechlorofluorocarbon. In other instances, dry powdered nutrients can besuspended, e.g., dispersed or dissolved, in aqueous solutions. In eithercase, when an aliquot of the nutrient suspension or solution is added tothe surface of the medium, the liquid can be allowed to evaporate,leaving ample nutrients along with the gelling agent. Additionally oralternatively, in any embodiment, one or more nutrient for growing yeastor mold microorganisms can be deposited into the culture device in anaqueous liquid (e.g., an aqueous suspending medium or diluent) when theculture device is inoculated.

The first and/or second hydrogel-forming composition (22 and 22′,respectively) of the present disclosure further may comprise at leastone nutrient to facilitate the growth of a yeast or mold microorganism.Yeast and mold microorganisms are metabolically and ecologically diverseand, thus, can utilize a variety of nutrients to support their growthand reproduction. Table 3 shows nonlimiting examples of genera thatinclude yeast and mold microorganisms according to the presentdisclosure.

TABLE 3 Exemplary yeast and mold genera. Yeast genera Mold generaCandida Alternaria Cryptococcus Aspergillus Debaryomyces BotrytisGalactomyces Cladosporium Hanseniaspora Colletotrichum IssatchenkiaFusarium Kluyveromyces Geotrichum Metschnikowia Monila Pichia MucorRhodotorula Penicillium Saccharomyces Pullularia SaccharomycodesRhizopus Schizosaccharomyces Thamnidium Sporobolomyces TrichotheciumTorulaspora Trichosporon Zygosaccharomyces

Culture media (e.g., dehydrated, powdered culture media) comprisingnutrients to facilitate the growth and reproduction of yeast and moldmicroorganisms are known in the art. Components of the culture mediainclude, for example, a source of nitrogen (e.g., yeast extract,enzymatic digests of meat or other proteins, malt extract); a source ofcarbon (e.g., various sugars, polysaccharides, oligosaccharides, maltextract); one or more various inorganic salts (e.g., calcium chloride,ferric ammonium citrate, magnesium sulfate, manganese chloride, zincsulfate); optionally, a buffering agent; and, optionally, an antibioticsuch as tetracycline or chloramphenicol, for example, to inhibit thegrowth of bacteria. In view of the present disclosure, a person havingordinary skill in the art will recognize a variety of nutrientcompositions that can be used with the enzyme substrate mixture of thepresent disclosure to detect yeast and mold microorganisms, provided acomponent of the nutrient composition does not substantially inhibit thehydrolysis of the enzyme substrates in the enzyme substrate mixture andprovided a component of the nutrient composition does not substantiallymask (e.g., by fluorescence quenching) the products of the enzymesubstrates in the enzyme substrate mixture.

In any embodiment, the culture device can comprise a nutrient thatincludes proteins, oligopeptides, and/or amino acids. Non-limitingexamples of such nutrients include yeast extract (e.g., yeastautolysate), malt extract, and peptic digest of meat.

In any embodiment, the culture device can comprise a carbohydratenutrient comprising a monosaccharide, a disaccharide, a trisaccharide,an oligosaccharide, or a combination of any two or more of theforegoing. Those skilled in the art will also recognize that varioussources of carbohydrates can be used. They can be natural sources (e.g.,potato or plant extracts), as mixtures of natural sources, in pure forms(such as oligosaccharides or monosaccharides), in mixtures of pureforms, or as mixture of pure and natural forms. The natural mixtures cancontain varying amounts of carbohydrates. Thus, carbohydrates may beprovided from a variety of sources. Malt extract is an exemplary sourceof a variety of carbohydrates. In addition to comprising proteins,oligopeptides, and/or amino acids that facilitate the growth andreproduction of yeast and mold microorganisms, malt extract alsocomprises carbohydrate nutrients that facilitate the growth andreproduction of yeast and mold microorganisms.

The natural mixtures can contain various types and amounts ofcarbohydrates, such as polysaccharides, oligosaccharides, andmonosaccharides. Polysaccharides that can be assimilated by yeasts andmolds include soluble starch or inulin. Oligosaccharides that can beassimilated by yeasts and molds are sucrose, maltose, cellobiose,trehalose, lactose, melibiose, raffinose, and melezitose.Monosaccharides that can be metabolized by yeasts and molds include thehexoses (six carbon sugars): e.g., D-glucose, D-fructose, D-galactose,D-mannose, L-rhamnose, and L-sorbose; as well as the pentoses (fivecarbon sugars): e.g., D-xylose, D-ribose, L-arabinose, and D-arabinose.

Not all carbohydrates must be provided and the relative amount of eachmay vary. Those in the art will recognize that many differentcombinations of monosaccharides can be used in the culture medium of thepresent disclosure. Normally, only the sugars that can be metabolized byyeasts and molds are provided.

For general guidance, specific amounts of carbohydrates are indicatedherein. These amounts are for general guidance only, and aredeterminable in accordance information known to those of skill in theart, and are not intended to be limiting. Those in the art willrecognize that many different combinations of carbohydrates can be usedin the culture device of the present disclosure. Normally, only thosesugars which can be utilized by any particular yeasts and molds to bedetected must be provided in the culture medium. Those skilled in theart will appreciate that other carbohydrates may be provided withoutdeparting from the invention.

In any embodiment, the culture device can comprise a buffering agent.The buffering agent can be provided in the first and/or secondhydrogel-forming composition. Nonlimiting examples of suitable bufferingagents include phosphate compounds (e.g., sodium dihydrogen phosphate,disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate), sodium carbonate, MOPS(2-[N-Morpholino]ethanesulfonic acid) free acid, and MOPS sodium salt.

The culture device of the present disclosure can comprise one or moreinorganic element to facilitate the growth of yeast and/or molds. Theseinclude any one or more of the following (to the extent not alreadyprovided in the above sources of various components of the culturemedium): calcium, chloride, cobalt, iron, manganese, phosphorus,potassium, sulfur, sodium, tin, and zinc. Salts may be provided as asource of ions. Salts may include (amounts per liter of medium):potassium phosphate, magnesium sulfate, sodium chloride, calciumchloride, boric acid, copper sulfate, potassium iodide, ferric chloride,manganese sulfate, sodium molybdate, and zinc sulfate. The inorganicelement(s) and/or salt(s) can be provided in the first powder and/orsecond powder, if present.

In any embodiment, a culture device of the present disclosure mayfurther comprise a selective agent. In order to grow a yeast or moldsample without interference from bacteria, bacteriostatic orbactericidal selective agents such as chloramphenicol,chlortetracycline, tartaric acid, or suitable penicillin, for example,can be included. The selective agent may be provided in the firsthydrogel-forming composition and/or second hydrogel-forming composition,if present. Alternatively or additionally, a selective agent may beprovided in the first adhesive composition and/or second adhesivecomposition, if present.

In another aspect, the present disclosure provides a method of detectinga yeast or mold microorganism. The method comprises contacting a sampleand an aqueous liquid with the gelling agent disposed on the first orsecond adhesive composition of any embodiment of the culture devicedisclosed herein. In any embodiment, the sample can comprise the aqueousliquid. In general, the amounts of gelling agent; indicator agents; andnutrient (if present) in the culture device are selected to provide aneffective concentration for detecting yeast or mold microorganisms whenthey are reconstituted with a predetermined volume (e.g., 1 milliliter,2 milliliters, 5 milliliters) of aqueous liquid. The aqueous liquid canbe added with the sample materials (e.g., the sample material can bedissolved, homogenized, suspended, and/or diluted in an aqueous liquidsuch as sterile water, an aqueous buffer, or an aqueous nutrient medium,for example). In any embodiment wherein the sample comprises solid(e.g., a membrane filter having retained material thereon or therein) orsemisolid materials, the predetermined volume of liquid (e.g., sterilewater, an aqueous nutrient medium) can be used to reconstitute theculture device before or after the solid or semisolid sample is used toinoculate the device.

The sample can be contacted with the gelling agent using methods thatare known in the art (e.g., by pouring or pipetting a liquid sample ontothe gelling agent). In an embodiment where the culture device comprisesa coversheet, the coversheet is typically lifted to permit deposition ofthe sample between the coversheet and the substrate; preferably, intothe aperture of a spacer, if present, in the culture device. In anyembodiment, contacting a sample and an aqueous liquid with the gellingagent forms an inoculated culture device. After forming the inoculatedculture device, the coversheet, if present, is lowered to form aprotective barrier against contamination and/or excess evaporation ofthe aqueous liquid during incubation. In any embodiment, the sample maybe spread evenly over the growth region, for example by placing aweighted plate on top of the covered device. If the coversheet is notpresent, preferably, the inoculated culture device is placed in asterile container to prevent contamination and/or excessive evaporationof the aqueous liquid during incubation.

The embodiment of device 11 illustrated in FIG. 3 is identical to thatof FIG. 2 except that spacer 24 is not present in FIG. 3. To use such anembodiment, a template (e.g., a weighted circular ring defining thegrowth region) can be applied temporarily on top of coversheet 18, afterclosing, to confine reconstitution of the medium to the growth region ofthe medium.

In any embodiment, contacting a sample with the gelling agent disposedon the first or second adhesive composition of the culture devicecomprises placing the sample in fluid communication with the at leastone nutrient. This can be achieved by suspending or diluting the samplein a liquid (e.g., an aqueous liquid) comprising a nutrient or bycontacting the sample and an aqueous liquid with a firsthydrogel-forming composition or second hydrogel-forming composition,described herein, comprising the at least one nutrient.

In any embodiment, the method further comprises incubating (e.g., in atemperature-controlled environmental chamber) the inoculated culturedevice for a period of time. The incubation conditions (e.g., theincubation temperature) can affect the rate of growth of yeast and moldmicroorganisms present in the sample. A person having ordinary skill inthe art will recognize suitable incubation temperatures to detectspecific yeast and mold microorganisms. An inoculated culture device ofthe present disclosure can be incubated, for example at temperaturesbetween about 20° C. to about 32° C., inclusive. In any embodiment, theculture device can be incubated under aerobic conditions.

The inoculated culture device is incubated for a period of timesufficient to permit the growth of a yeast or mold microorganism. In anyembodiment, the period of time can be about 36 hours to about 120 hours,inclusive. In any embodiment, the period of time can be about 48 hoursto about 120 hours, inclusive. In any embodiment, the period of time canbe about 48 hours to about 96 hours, inclusive. In any embodiment, theperiod of time can be about 48 hours to about 72 hours, inclusive. Inany embodiment, the period of time can be about 48 hours to about 48hours, inclusive. In any embodiment, the period of time can be up toabout 72 hours. In any embodiment, the period of time can be up to about60 hours. In any embodiment, the period of time can be up to about 48hours.

The method of the present disclosure further comprises detecting a yeastor mold colony in the culture device (e.g., observing a yeast or moldcolony in the culture device). In any embodiment, detecting a yeast ormold colony in the culture device can comprise detecting in the culturedevice a presence or an absence of the detectable reporter group of atleast one of the indicator agents, wherein detecting the presence of thedetectable reporter group is indicative of a presence of a colony ofyeast or mold microorganisms. As a yeast or mold colony grows in theculture device of the present disclosure, the cells in the colony reactwith one or more of the indicator agents to activate (e.g., byhydrolysis of a chromogenic or fluorogenic enzyme substrate) thereporter group thereby directly or indirectly making the reporter groupdetectable. In the case of chromogenic indicator agents, the reportergroup can be detected by the characteristic wavelengths of light that itabsorbs and/or reflects. For example, indicator agents comprising anindolyl reporter group can dimerize to form indigo or derivativesthereof. Thus, the presence of a colony having a color (e.g., either thecolony having the color or the hydrogel proximate the colony having thecolor) that is associated with a particular reporter group is indicativeof a colony of yeast or mold microorganisms.

In the case of fluorogenic indicator agents, the detectable reportergroup can be observed by illuminating the culture device with anappropriate wavelength of light (e.g., about 365 nm to detect a reportergroup comprising 4-methylumbelliferone) and observing the light emittedby the reporter group. A person having ordinary skill in the art willrecognize suitable wavelengths of light required respectively toilluminate the culture device and to detect a reporter group associatedwith a particular fluorogenic indicator agent. A colony having the colorof the fluorescent reporter group or the presence of the fluorescentreporter group in the hydrogel proximate the colony is in indication thecolony is a yeast colony or a mold colony.

In any embodiment, detecting the reporter group can comprise observingthe culture device visually. In any embodiment, detecting the reportergroup can comprise obtaining an image of the culture device andobserving the image visually or analyzing the image using automatedimage-analysis techniques. Methods and devices for automated detectionof microbial colonies in a culture device are described, for example, inU.S. Pat. Nos. 5,448,652; 6,058,209; 6,243,486; 6,271,022; 7,298,885;and 7,319,031; which are all incorporated herein by reference in theirentirety.

In any embodiment, the method of the present disclosure furthercomprises enumerating yeast or mold colonies present in the inoculatedculture device after incubating the inoculated culture device. Thus,after the yeast or mold colonies are detected as described herein, thenumber of detected colonies is determined either manually or usingautomated processes known in the art.

EMBODIMENTS

Embodiment A is a culture device, comprising:

a body comprising a self-supporting substrate having a first majorsurface and a second major surface;

a first adhesive composition disposed on a portion of the first majorsurface of the substrate;

a substantially dry, cold-water-soluble first hydrogel-formingcomposition adhered to the first adhesive composition; and

a plurality of indicator agents, the plurality of indicator agentscomprising:

-   -   three indicator agents for detecting distinct glycosidase enzyme        activities;    -   an indicator agent for detecting an alkyl esterase enzyme        activity;    -   an indicator agent for detecting a phosphatase enzyme activity;

wherein each of the plurality of indicator agents comprises a detectablereporter group.

Embodiment B is the culture device of Embodiment A, further comprising acover sheet attached to the body member.

Embodiment C is the culture device of Embodiment B, wherein the coversheet comprises a first major surface facing the body member; whereinthe culture device further comprises a second adhesive compositiondisposed on a portion of the first major surface of the cover sheet anda substantially dry, cold-water-soluble second hydrogel-formingcomposition adhered to the second adhesive composition.

Embodiment D is the culture device of any one of the precedingEmbodiments, wherein at least one of the plurality of indicator agentsis disposed in the first adhesive composition, the second adhesivecomposition, the first hydrogel-forming composition, and/or the secondhydrogel-forming composition.

Embodiment E is the culture device of Embodiment D, wherein at leastthree of the plurality of indicator agents are disposed in the firstadhesive composition and/or the second adhesive composition.

Embodiment F is the culture device of Embodiment E, wherein at leastfive of the indicator agents are disposed in the first adhesivecomposition and/or the second adhesive composition.

Embodiment G is the culture device of any one of the precedingEmbodiments, further comprising an air-permeable membrane disposedbetween the substrate and the first hydrogel-forming composition.

Embodiment H is the culture device of any one of the precedingEmbodiments, wherein the at least three indicator agents for detectingdistinct glycosidase enzyme activities include a compound to detectalpha-glucosidase enzyme activity, a compound to detect beta-glucosidaseenzyme activity, and a compound to detect beta-galactosidase enzymeactivity.

Embodiment I is the culture device of any one of the precedingEmbodiments, wherein at least one of the plurality of indicator agentsis a chromogenic enzyme substrate or fluorogenic enzyme substrate.

Embodiment J is the culture device of Embodiment I, wherein each of theplurality of indicator agents is a chromogenic enzyme substrate orfluorogenic enzyme substrate.

Embodiment K is the culture device of any one of the precedingEmbodiments, wherein the at least three indicator agents for detectingdistinct glycosidase enzyme activities comprise5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside, and5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside.

Embodiment L is the culture device of any one of the precedingEmbodiments, wherein the indicator agent for detecting an alkyl esteraseenzyme activity comprises 3-indolyl-acetate.

Embodiment M is the culture device of Embodiment L, wherein theindicator agent for detecting an alkyl esterase enzyme activitycomprises 5-bromo-4-chloro-3-indolyl-acetate.

Embodiment N is the culture device of any one of the precedingEmbodiments, wherein the indicator agent for detecting a phosphataseenzyme activity comprises 5-bromo-4-chloro-3-indolyl-phosphate.

Embodiment O is the culture device of any one of the precedingEmbodiments, further comprising a water-insoluble spacer having anaperture, the spacer being attached to the body member or the coversheetand the entire aperture being positioned between the body member and thecover sheet.

Embodiment P is the culture device of any one of the precedingEmbodiments, further comprising a predefined volume of aqueous liquiddisposed between the body member and the coversheet, wherein the aqueousliquid and gelling agent form a hydrogel.

Embodiment Q is the culture device of Embodiment P, wherein the at leastone nutrient comprises malt extract.

Embodiment R is the culture device of Embodiment P or Embodiment Q,wherein the at least one nutrient is selected from the group consistingof a peptic digest of meat, yeast extract, dextrose, potassiumphosphate, ferric ammonium citrate, magnesium sulfate, manganesechloride, sodium carbonate, zinc sulfate, or a combination of any two ormore of the foregoing nutrients.

Embodiment S is the culture device of any one of the precedingEmbodiments, wherein the first hydrogel-forming composition or thesecond hydrogel-forming composition comprises substantially dryagglomerated powders.

Embodiment T is the culture device of Embodiment S, wherein at least oneof the plurality of indicator agents is disposed in the second adhesivecomposition.

Embodiment U is the culture device of Embodiment O as dependent onEmbodiment N, wherein the spacer comprises a perimeter that defines agrowth area and a thickness dimension, wherein the thickness dimensionis selected to prevent contact between hydrogel and the coversheet inthe growth area.

Embodiment V is the culture device of any one of the precedingEmbodiments, further comprising a selective agent disposed in the firstadhesive composition or the second adhesive composition.

Embodiment W is a method of detecting yeast and mold in a sample,comprising:

contacting a sample and an aqueous liquid with the gelling agentdisposed on the first or second adhesive composition of the culturedevice of any one of Embodiments A through V to form an inoculatedculture device;

incubating the inoculated culture device for a period of time; and

detecting a yeast or mold colony in the culture device.

Embodiment X is the method of Embodiment W, wherein detecting a yeast ormold colony in the culture device comprises detecting in the culturedevice a presence or an absence of the detectable reporter group of atleast one of the indicator agents, wherein detecting the presence of thedetectable reporter group is indicative of a presence of a colony ofyeast or mold microorganisms.

Embodiment Y is the method of Embodiment W or Embodiment X, whereincontacting a sample with the gelling agent disposed on the first orsecond adhesive composition of the culture device comprises placing thesample in fluid communication with the at least one nutrient.

Embodiment Z is the method of any one of Embodiments W through Y,wherein incubating the inoculated culture device comprises incubatingthe inoculated culture device at a temperature between about 20° C. andabout 32° C., inclusive.

Embodiment AA is the method of any one of Embodiments W through Z,wherein incubating the inoculated culture device for a period of timecomprises incubating the inoculated culture device for up to about 72hours.

Embodiment BB is the method of Embodiment AA, wherein incubating theinoculated culture device for a period of time comprises incubating theinoculated culture device for up to about 60 hours.

Embodiment CC is the method of Embodiment BB, wherein incubating theinoculated culture device for a period of time comprises incubating theinoculated culture device for up to about 48 hours.

Embodiment DD is the method of any one of Embodiments W through CC,further comprising enumerating yeast or mold colonies present in theinoculated culture device after incubating the inoculated culturedevice.

Advantages and embodiments of this disclosure are further illustrated bythe following examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this disclosure. All materialsare commercially available or known to those skilled in the art unlessotherwise stated or apparent.

EXAMPLES

Indicators

The chromogenic substrate indicators that were used in the examples arelisted in Table 4 and were purchased from Biosynth International, Inc.(Itasca, Ill.).

TABLE 4 Indicator Name CAS Number 5-Bromo-4-chloro-3-indolyl acetate(X-3-Acetate) 3252-36-65-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 7240-90-6 (X-Gal)5-Bromo-4-chloro-3-indolyl-beta-D-glucopyranoside 15548-60-4 (X-b-Glu)5-Bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside 108789-36-2 (X-a-Glu)5-Bromo-4-chloro-3-indolyl phosphate para-toluidine salt 6578-06-9 (BCIPp-toluidine)

Each of the Indicator Coating Formulations A-D (Table 5) was preparedfrom the five chromogenic substrate indicators in the amounts specifiedin Table 5 and 100 g of a noninhibitory adhesive copolymer of isooctylacrylate and acrylamide (described below).

TABLE 5 Indicator Coating Formulations A-D: Chromogenic SubstrateIndicator Composition (mg) of the Indicator Coating Formulation A B C DX-3-Acetate 43.3 57.7 57.7 121.2 X-Gal 81.8 81.8 81.8 171.8 X-b-Glu 81.881.8 81.8 171.8 X-a-Glu 81.8 81.8 81.8 171.8 BCIP p-toluidine 173.3130.0 86.7 182.0

Culture Media Formulations

Six separate culture media formulations were prepared as homogeneousmixtures by blending the formulation ingredients together. Thecompositions of Culture Media Formulations E-J are described in Tables6-11.

TABLE 6 Culture Media Formulation E Amount Ingredient (grams) SourcePeptic digest of animal 6.3 g Alpha Biosciences (Baltimore, MD) tissueMagnesium sulfate 0.6 g EMD Millipore (Billerica, MA) Ferric ammonium0.3 g Sigma-Aldrich (St. Louis, MO) citrate Calcium chloride 0.1 gMallinckrodt (St. Louis, MO) Potassium phosphate 1.3 g EMD Millipore(Billerica, MA) monobasic Sodium carbonate 0.2 g Sigma-Aldrich (St.Louis, MO) Dextrose 11.3 g  Becton, Dickinson (New Franklin, NJ) TotalIngredients 20.1 g 

TABLE 7 Culture Media Formulation F Amount Ingredient (grams) SourcePeptic digest of animal 7.1 g Alpha Biosciences (Baltimore, MD) tissueYeast extract 4.3 g Alpha Biosciences (Baltimore, MD) Magnesium sulfate0.7 g EMD Millipore (Billerica, MA) Ferric ammonium 0.4 g Sigma-Aldrich(St. Louis, MO) citrate Calcium chloride 0.1 g Mallinckrodt (St. Louis,MO) Sodium carbonate 0.2 g Sigma-Aldrich (St. Louis, MO) Dextrose 7.1 gBecton, Dickinson (New Franklin, NJ) Total Ingredients 19.9 g 

TABLE 8 Culture Media Formulation G Amount Ingredient (grams) SourcePeptic digest of animal 6.6 g Alpha Biosciences (Baltimore, MD) tissueMalt extract 4.9 g EMD Millipore (Billerica, MA) Yeast extract 1.6 gAlpha Biosciences (Baltimore, MD) Magnesium sulfate 1.0 g EMD Millipore(Billerica, MA) Ferric ammonium 0.6 g Sigma-Aldrich (St. Louis, MO)citrate Calcium chloride 0.1 g Mallinckrodt (St. Louis, MO) Zinc sulfate0.7 g EMD Millipore (Billerica, MA) Potassium phosphate 0.5 g EMDMillipore (Billerica, MA) monobasic Sodium carbonate 0.2 g Sigma-Aldrich(St. Louis, MO) Dextrose 3.9 g Becton, Dickinson (New Franklin, NJ)Total Ingredients 20.1 g 

TABLE 9 Culture Media Formulation H Amount Ingredient (grams) SourcePeptic digest of animal 7.0 g Alpha Biosciences (Baltimore, MD) tissueMalt extract 4.5 g EMD Millipore (Billerica, MA) Calcium chloride 0.1 gMallinckrodt (St. Louis, MO) Manganese chloride 0.1 g Alfa Aesar (WardHill, MA) Yeast extract 2.5 g Alpha Biosciences (Baltimore, MD)Magnesium sulfate 0.1 g EMD Millipore (Billerica, MA) Ferric ammonium0.2 g Sigma-Aldrich (St. Louis, MO) citrate Zinc sulfate 0.1 g EMDMillipore (Billerica, MA) Potassium phosphate 0.5 g EMD Millipore(Billerica, MA) monobasic Sodium carbonate 0.1 g Sigma-Aldrich (St.Louis, MO) Dextrose 5.0 g Becton, Dickinson (New Franklin, NJ) TotalIngredients 20.2 g 

TABLE 10 Culture Media Formulation I Amount Ingredient (grams) SourceProteose peptone 5.0 g Becton, Dickinson (New Franklin, NJ) Casein 3.1 gBecton, Dickinson (New Franklin, NJ) Peptic casein digest 1.2 g Becton,Dickinson (New Franklin, NJ) Yeast extract 1.9 g Alpha Biosciences(Baltimore, MD) Ferric ammonium 0.2 g Sigma-Aldrich (St. Louis, MO)citrate Calcium chloride 0.1 g Mallinckrodt (St. Louis, MO) Sodiumcarbonate 0.1 g Sigma-Aldrich (St. Louis, MO) Dextrose 8.5 g Becton,Dickinson (New Franklin, NJ) Total Ingredients 20.1 g 

TABLE 11 Culture Media Formulation J Amount Ingredient (grams) SourceMalt extract 20.0 g EMD Millipore (Billerica, MA) Total Ingredients 20.0g

Inoculation and Incubation

With the exception of S. cerevisiae, H. anomala, and C. albicans, theyeast and mold strains that were used in the examples (and listed inTable 12) were purchased as lyophilized discs from MicroBiologics, Inc.(St. Cloud, Minn.). The strains of S. cerevisiae, H. anomala, and C.albicans were purchased as KWIK STIK devices from MicroBiologics, Inc.,and were propagated according to the manufacturer's instructions. Asingle lyophilized disc of each strain was separately added to 10 to 30milliliters of 0.1% peptone water (Catalog No. D299, Hardy Diagnostics,Santa Maria, Calif.) to resuspend the organisms from the lyophilizeddisc material. The samples were each serially diluted in 0.1% peptonewater to yield concentrations that provided counts of colony formingunits (cfu) within the counting range of the thin film culture device(approximately 15-150 cfu per device).

Dichloran Rose Bengal Chloramphenicol (DRBC) Agar Petri dish plates wereprepared as reference culturing devices (Becton Dickinson Company,Franklin Lakes, N.J.). A 100 microliter aliquot of inoculum was spreadplated onto the DRBC plate and the plate was incubated at 25° C. for 5days. The counts obtained with the DRBC reference plate were multipliedby a dilution factor of 10 to account for the differences in inoculumvolume added to the DRBC reference plates and the experimental thin filmculture plates.

TABLE 12 Yeast and Mold Strains Paecilomyces spp. (3M-M10) Saccharomycescerevisiae (ATCC 7754) Botrytis spp. (3M-M97) Hansenula anomala (3M-Y28)Candida albicans (ATCC 10231) Penicillium chrysogenum (ATCC 10106)Trichosporon mucoides (ATCC 201382) Candida guilliermondii (ATCC 6260)Aspergillus niger (3M-M6)

Example 1

The Indicator Coating Formulation B (Table 5) was prepared by mixing thefive chromogenic substrate indicators in DMSO (5-15 mL) and then addingto 100 g of the noninhibitory adhesive copolymer (approximately 24%solids in a solution in ethyl acetate and heptanes) described in Example1 of U.S. Pat. No. 5,635,367, which is incorporated herein by referencein its entirety. The resulting coating formulation was stirred untilhomogeneous. The formulation was knife coated (gap setting of about 1mil) onto one side of a 1.6 mil thick bi-axially oriented polypropylene(BOPP) film substrate (Vifan Company, Morristown, Tenn.). The coatedfilm was dried in an oven at 70-80° C. for 10-15 minutes. The coatedside of the BOPP film was then powder coated with a 1:1 mixture ofxanthan gum and locust bean gum. The excess powder was shaken loose.

The thin film culture device for the experiment was prepared by removingthe thin film cover sheet from a commercially available 3M PETRIFILMYeast & Mold Count Pate (3M Company) and replacing it with the coatedBOPP film described above. The coated BOPP film was cut to the samedimensions as the bottom film and was it was oriented so that the coatedsurface of the film faced the culture medium. The new thin film coversheet containing the five indicators was attached to the bottom filmusing double-sided tape.

The thin film culture devices were inoculated with a single yeast ormold sample selected from Paecilomyces spp., Saccharomyces cerevisiae,Botrytis spp., Hansenula anomala, Candida albicans, Penicilliumchrysogenum. The top film of the device was lifted up and 1 mL of theinoculum was added (by pipet) to the culture medium on the bottom film.The top film was replaced and the sample was uniformly distributed tothe desired area (30 mm²) using a 3M PETRIFILM Flat Spreader (3MCompany). Duplicate thin film culture devices were prepared for eachsample. The inoculated devices were incubated at 25° C. for 60 to 72hours.

The colonies in each device were counted by visual examination at 48hours and at the end of the incubation period (60-72 hour timepoint). Ateach timepoint, the cfu counts of the individual devices were averagedand the average count value was determined. The colonies on referenceDRBC plates were counted in the same manner as described for the thinfilm culture devices. The results for the 48 hour time point arepresented in Tables 13 and 14 together with the results obtained forComparative Examples 1-6 and for the DRBC reference plates. The cfucounts for the DRBC plates were taken after 5 days of incubation.

Comparative Example 1

The same procedure as described in Example 1 was used with the exceptionthat only a single indicator agent, 5-bromo-4-chloro-3-indolyl acetate(57.7 mg per 100 g of adhesive), was included in the top film coating(instead of five indicator agents). For each yeast or mold sample, thedevice was inoculated with the same sample preparation as used inExample 1. The results are presented in Tables 13 and 14.

Comparative Example 2

The same procedure as described in Example 1 was used with the exceptionthat only a single indicator agent,5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (81.8 mg per 100 gof adhesive), was included in the top film coating (instead of fiveindicator agents). For each yeast or mold sample, the device wasinoculated with the same sample preparation as used in Example 1. Theresults are presented in Tables 13 and 14.

Comparative Example 3

The same procedure as described in Example 1 was used with the exceptionthat only a single indicator agent,5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside (81.8 mg per 100 g ofadhesive), was included in the top film coating (instead of fiveindicator agents). For each yeast or mold sample, the device wasinoculated with the same sample preparation as used in Example 1. Theresults are presented in Tables 13 and 14.

Comparative Example 4

The same procedure as described in Example 1 was used with the exceptionthat only a single indicator agent,5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside (81.8 mg per 100 g ofadhesive), was included in the top film coating (instead of fiveindicator agents). For each yeast or mold sample, the device wasinoculated with the same sample preparation as used in Example 1. Theresults are presented in Tables 13 and 14.

Comparative Example 5

The same procedure as described in Example 1 was used with the exceptionthat only a single indicator agent, 5-bromo-4-chloro-3-indolyl phosphatepara-toluidine salt (130 mg per 100 g of adhesive), was included in thetop film coating (instead of five indicator agents). For each yeast ormold sample, the device was inoculated with the same sample preparationas used in Example 1. The results are presented in Tables 13 and 14.

TABLE 13 Average Colony Count (cfu) Paecilomyces Penicillium spp.chrysogenum Botrytis spp. Example 1 73 247 60 Comparative Example 1 35250 0 Comparative Example 2 50 0 0 Comparative Example 3 0 0 0Comparative Example 4 0 0 0 Comparative Example 5 0 0 0 ReferenceExample 50 220 50 (DRBC agar)

TABLE 14 Average Colony Count (cfu) Hansenula Candida Saccharomycesanomala albicans cerevisiae Example 1 491 430 90 Comparative Example 1301 251 60 Comparative Example 2 0 0 0 Comparative Example 3 427 0 0Comparative Example 4 416 410 60 Comparative Example 5 384 0 0 ReferenceExample 280 460 35 (DRBC agar)

Example 2

The thin film culture devices were prepared using the general proceduresdescribed in U.S. Pat. Nos. 4,565,783 and 5,089,413. The coatingformulation was prepared by adding a solution containing5-bromo-4-chloro-3-indolyl phosphate para-toluidine salt (86.7 mg),chlortetracycline (13.6 mg), and chloramphenicol (10.9 mg) in methanol(5-10 mL) to 100 g of the acrylate copolymer adhesive described inExample 1. The resulting coating formulation was stirred untilhomogeneous. The coating substrate was a microporous polyolefin film(Tredegar EXXAIRE film, Tredegar Film Products, Richmond, Va.)adhesively laminated to polysilk paper. The coating formulation wasknife coated (gap setting of about 1 mil) onto the microporouspolyolefin film side of the substrate to create a master roll. Themaster roll was cut into 76 mm wide by 102 mm long sections that formedthe bottom layer of the device. A foam layer (76 mm wide by 102 mm longby 0.57 mm thick) with a circular opening (61 mm in diameter) wasadhesively laminated to the coated side of the bottom film. The circularopening was positioned in the center of the foam layer.

The culture medium was prepared by adding a mixture of xanthan gum andlocust bean gum (1:1 by weight) to the pre-blended Culture MediaFormulation E (described in Table 6) in a ratio of three parts gummixture to one part Culture Media Formulation E. The combined mixturewas blended to complete the mixing. Approximately two grams of thecombined powder mixture was evenly applied to cover the adhesive exposedby the circular opening in the thin film culture device. Excess powderwas removed by inverting the device. The result was a thin layer of thecombined powder mixture (culture medium and gum) bonded to the adhesivelayer of the device.

The thin film cover sheet containing Indicator Coating Formulation B wasprepared according to the procedure described in Example 1. The coatedcover sheet was cut to the same dimensions as the bottom film sectionand was it was oriented so that the coated surface of the cover sheetfaced the culture medium. The cover sheet was attached to the bottomfilm using double-sided tape.

The thin film culture devices were inoculated with a single yeast sampleselected from Candida albicans, Trichosporon mucoides, Candidaguilliermondii. The top film of the device was lifted up and 1 mL of theinoculum was added (by pipet) to the culture medium on the bottom film.The top film was replaced and the sample was uniformly spread to theedges of the circular opening by applying downward pressure with a 3MPETRIFILM Flat Spreader. Duplicate thin film culture devices wereprepared for each sample. The inoculated devices were incubated at 25°C. for 60 to 72 hours.

The colonies from each sample were counted by visual examination at 48hours and at the end of the incubation period (60-72 hour timepoint).The colonies were either white or blue in color. At each timepoint, thecfu counts of the individual devices were averaged and the average countvalue was used to calculate the number of colony forming units permilliliter (cfu/mL) in the original undiluted sample. The calculatedcfu/mL values and colony color (white or blue) for each sample at the 48hour timepoint are reported in Table 15 together with the resultsobtained for Examples 3-6.

Example 3

The same experimental conditions and procedures as described in Example2 were followed with the exception that the thin film culture deviceswere prepared using Culture Media Formulation F (described in Table 7),instead of Culture Media Formulation E (described in Table 6).

Example 4

The same experimental conditions and procedures as described in Example2 were followed with the exception that the thin film culture deviceswere prepared using Culture Media Formulation G (described in Table 8),instead of Culture Media Formulation E (described in Table 6).

Example 5

The same experimental conditions and procedures as described in Example2 were followed with the exception that the thin film culture deviceswere prepared using Culture Media Formulation H (described in Table 9),instead of Culture Media Formulation E (described in Table 6).

Example 6

The same experimental conditions and procedures as described in Example2 were followed with the exception that the thin film culture deviceswere prepared using Culture Media Formulation I (described in Table 10),instead of Culture Media Formulation E (described in Table 6).

Example 7

The same experimental conditions and procedures as described in Example2 were followed with the exception that the thin film culture deviceswere prepared using Culture Media Formulation J (described in Table 11),instead of Culture Media Formulation E (described in Table 6).

TABLE 15 Culture cfu/mL (colony color) Media Candida TrichosporonCandida Formulation albicans mucoides guilliermondii Example 2 E 2.1 ×10⁷ (blue) 9.6 × 10⁵ (blue) 1.6 × 10⁷ (white) Example 3 F 2.0 × 10⁷(blue) 1.1 × 10⁶ (blue) 1.8 × 10⁷ (white) Example 4 G 2.2 × 10⁷ (blue)7.2 × 10⁵ (blue) 1.6 × 10⁷ (blue) Example 5 H 2.0 × 10⁷ (blue) 7.6 × 10⁵(blue) 1.0 × 10⁷ (blue) Example 6 I 3.3 × 10⁷ (white) 7.2 × 10⁵ (white)1.7 × 10⁷ (white) Example 7 J NT 8.5 × 10⁵ (blue) 2.0 × 10⁷ (light blue)

Example 8

The thin film culture devices as described in Example 2 were preparedwith two exceptions. First, the thin film culture devices were preparedusing Culture Media Formulation H (described in Table 9), instead ofCulture Media Formulation E (described in Table 6). Second, the thinfilm cover sheet of the device was coated with Indicator CoatingFormulation A, instead of Indicator Coating Formulation B.

The thin film culture devices were inoculated with a single yeast ormold sample selected from Paecilomyces spp., Saccharomyces cerevisiae,Botrytis spp., Hansenula anomala, Candida albicans, Penicilliumchrysogenum, Aspergillus niger. The top film of the device was lifted upand 1 mL of the inoculum was added (by pipet) to the culture medium onthe bottom film. The top film was replaced and the sample was uniformlyspread to the edges of the circular opening by applying downwardpressure with a 3M PETRIFILM Flat Spreader. Duplicate thin film culturedevices were prepared for each sample. The inoculated devices wereincubated at 25° C. for 48 to 60 hours.

The colonies from each sample were counted by visual examination ateither the 48 hour or 60 hour timepoint. The cfu counts of theindividual devices were averaged and the average count value was used tocalculate the number of colony forming units per milliliter (cfu/mL) inthe original undiluted sample. In Tables 16 and 17, the calculatedcfu/mL values for each sample at either the 48 hour or 60 hour timepointare presented.

Example 9

The same experimental conditions and procedures as described in Example8 were followed with the exception that the thin film cover sheet of thedevice was coated with Indicator Coating Formulation B, instead ofIndicator Coating Formulation A. The results are presented in Tables 16and 17.

Example 10

The same experimental conditions and procedures as described in Example8 were followed with the exception that the thin film cover sheet of thedevice was coated with Indicator Coating Formulation C, instead ofIndicator Coating Formulation A. The results are presented in Tables 16and 17.

TABLE 16 Colony counts after 48 hours of incubation Indicator cfu/mLCoating Hansenula Saccharomyces Candida Formulation anomala cerevisiaealbicans Example 8 A 6.5 × 10⁷ 1.1 × 10⁷ 3.9 × 10⁷ Example 9 B 4.7 × 10⁶2.5 × 10⁶ 1.1 × 10⁸ Example 10 C 1.5 × 10⁶ 9.4 × 10⁵ 2.4 × 10⁵

TABLE 17 Colony counts after 48 or 60 hours of incubation (asdesignated). Indicator cfu/mL Coating Paecilomyces AspergillusPenicillium Formulation spp. Botrytis spp. niger chrysogenum Example 8 A6.5 × 10⁴ (a) 4.6 × 10⁴ (b) 2.0 × 10⁴ (b) 4.5 × 10⁶ (a) Example 9 B 8.4× 10⁴ (a) 5.2 × 10⁴ (a) 2.8 × 10⁴ (a) 3.1 × 10⁶ (b) Example 10 C 6.6 ×10⁴ (a) 6.0 × 10⁴ (a) 3.3 × 10⁴ (a) 3.4 × 10⁶ (a) (a) Colony countedafter 48 hours of incubation. (b) Colony counted after 60 hours ofincubation.

Example 11

The same thin film culture devices as described in Example 8 wereprepared with the exception that the thin film cover sheet of the devicewas coated with Indicator Coating Formulation D, instead of IndicatorCoating Formulation A.

Six food items were selected for testing (corn kernels, cut carrots,tomato paste, green chili puree, pasta in meat sauce, and chickenseasoning). Each food item was tested separately. An 11 g portion of thefood sample was added to a plastic enrichment bag containing 99 mL of0.1% peptone water and the bag was shaken. The food samples wereserially diluted with 0.1% peptone water to yield concentrations thatprovided counts of colony forming units (cfu) within the counting rangeof the thin film culture device (approximately 15-100 cfu per device).The thin film devices were inoculated by lifting the transparent filmcover sheet, pipetting 1 mL of the diluted sample in the center of thecoated bottom film, and replacing the cover sheet. The sample wasuniformly spread to the edges of the circular opening by applyingdownward pressure with a 3M PETRIFILM Flat Spreader. Duplicate thin filmculture devices were prepared for each sample. The inoculated deviceswere incubated at 25° C. for 60 to 72 hours.

The total number of yeast and mold colonies from each sample was countedby visual examination at 48 hours and at the end of the incubationperiod (60-72 hour timepoint). At each timepoint, the cfu counts of theindividual devices were averaged and the average count value was used tocalculate the number of colony forming units per milliliter (cfu/mL) inthe original undiluted sample. The colonies on reference DRBC plateswere counted in the same manner as described for the thin film culturedevices. The calculated cfu/mL values for each sample at the 48 hourtimepoint are presented in Table 18. The cfu counts for the DRBCreference plates were taken after 5 days of incubation.

TABLE 18 cfu/mL Using Thin Film cfu/mL Using DRBC Agar Food SampleDevice of Example 11 (Reference Example) Corn kernels 1.5 × 10⁶ 7.8 ×10⁶ Cut carrots 8.9 × 10⁶ 2.5 × 10⁷ Tomato paste 1.8 × 10⁴ 2.3 × 10³Green Chili Puree 7.5 × 10⁷ 7.0 × 10⁷ Pasta in Meat 2.1 × 10⁶ 2.5 × 10⁶Sauce Chicken 6.0 × 10³ 8.0 × 10³ Seasoning

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Various modifications may be made without departing from the spirit andscope of the invention. These and other embodiments are within the scopeof the following claims.

The invention claimed is:
 1. A culture device, comprising: a bodycomprising a self-supporting substrate having a first major surface anda second major surface; a first adhesive composition disposed on aportion of the first major surface of the substrate; a substantiallydry, cold-water-soluble first hydrogel-forming composition adhered tothe first adhesive composition; and a plurality of indicator agents, theplurality of indicator agents comprising: 5-bromo-4-chloro-3-indolylacetate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside, and5-bromo-4-chloro-3-indolyl phosphate para-toluidine salt; and a coversheet attached to the body member, wherein the cover sheet comprises afirst major surface facing the body member, and wherein the culturedevice also comprises a second adhesive composition disposed on aportion of the first major surface of the cover sheet and asubstantially dry, cold-water-soluble second hydrogel-formingcomposition adhered to the second adhesive composition; wherein at leastthree of the 5-bromo-4-chloro-3-indolyl acetate,5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside, and5-bromo-4-chloro-3-indolyl phosphate para-toluidine salt are disposed inthe first adhesive composition and/or the second adhesive composition.2. The culture device of claim 1, wherein the 5-bromo-4-chloro-3-indolylacetate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside, and5-bromo-4-chloro-3-indolyl phosphate para-toluidine salt are disposed inthe first adhesive composition and/or the second adhesive composition.3. The culture device of claim 1, further comprising a water-insolublespacer having an aperture, the spacer being attached to the body memberor the coversheet and the entire aperture being positioned between thebody member and the cover sheet.
 4. The culture device of embodiment 21,further comprising a predefined volume of aqueous liquid disposedbetween the body member and the coversheet, wherein the aqueous liquidand gelling agent form a hydrogel.
 5. A method of detecting yeast ormold in a sample, comprising: contacting a sample and an aqueous liquidwith the gelling agent disposed on the first or second adhesivecomposition of the culture device of claim 1 to form an inoculatedculture device; incubating the inoculated culture device for a period oftime; and detecting a yeast or mold colony in the culture device.
 6. Themethod of claim 5, wherein detecting a yeast or mold colony in theculture device comprises detecting in the culture device a presence oran absence of the detectable reporter group of at least one of theindicator agents, wherein detecting the presence of the detectablereporter group is indicative of a presence of a colony of yeast or moldmicroorganisms.
 7. The method of claim 5, wherein contacting a samplewith the gelling agent disposed on the first or second adhesivecomposition of the culture device comprises placing the sample in fluidcommunication with the at least one nutrient.
 8. The method of claim 5,wherein incubating the inoculated culture device comprises incubatingthe inoculated culture device at a temperature between about 20° C. andabout 32° C., inclusive.
 9. The method of claim 5, wherein incubatingthe inoculated culture device for a period of time comprises incubatingthe inoculated culture device for up to about 72 hours.
 10. The methodof claim 8, wherein incubating the inoculated culture device for aperiod of time comprises incubating the inoculated culture device for upto about 60 hours.
 11. The method of claim 5, further comprisingenumerating yeast or mold colonies present in the inoculated culturedevice after incubating the inoculated culture device.